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Background: Elevated serum ferritin is a common condition in critically ill patients. It is well known that hyperferritinemia constitutes a good biomarker for hemophagocytic lymphohistiocytosis (HLH) in critically ill patients. However, further differential diagnoses of hyperferritinemia in adult critically ill patients remain poorly investigated. We sought to systematically investigate hyperferritinemia in adult critically ill patients without HLH. Methods: In this secondary analysis of a retrospective observational study, patients ≥18 years admitted to at least one adult intensive care unit at Charité–Universitätsmedizin Berlin between January 2006 and August 2018, and with hyperferritinemia of ≥500 μg/L were included. Patients with HLH were excluded. All patients were categorized into non-sepsis, sepsis, and septic shock. They were also classified into 17 disease groups, based on their ICD-10 codes, and pre-existing immunosuppression was determined. Uni- and multivariable linear regression analyses were performed in all patients. Results: A total of 2583 patients were analyzed. Multivariable linear regression analysis revealed positive associations of maximum SOFA score, sepsis or septic shock, liver disease (except hepatitis), and hematological malignancy with maximum ferritin. T/NK cell lymphoma, acute myeloblastic leukemia, Kaposi’s sarcoma, acute or subacute liver failure, and hepatic veno-occlusive disease were positively associated with maximum ferritin in post-hoc multivariable linear regression analysis. Conclusions: Sepsis or septic shock, liver disease (except hepatitis) and hematological malignancy are important differential diagnoses in hyperferritinemic adult critically ill patients without HLH. Together with HLH, they complete the quartet of important differential diagnoses of hyperferritinemia in adult critically ill patients. As these conditions are also related to HLH, it is important to apply HLH-2004 criteria for exclusion of HLH in hyperferritinemic patients. Hyperferritinemic critically ill patients without HLH require quick investigation of differential diagnoses.

OBJECTIVE: Hemophagocytic lymphohistiocytosis is a hyperinflammatory syndrome that often requires critical care support and remains difficult to diagnose. These guidelines are meant to aid in the early recognition, diagnosis, supportive care, and treatment of patients with hemophagocytic lymphohistiocytosis in ICUs. DATA SOURCES: The literature searches were performed with PubMed (MEDLINE). STUDY SELECTION: Keywords and medical subject headings terms for literature search included "macrophage activation syndrome," hemophagocytic lymphohistiocytosis," and "hemophagocytic syndrome." DATA EXTRACTION: The Histiocyte Society developed these consensus recommendations on the basis of published reports and expert opinions with level of evidence provided for each recommendation. They were endorsed by the Society of Critical Care Medicine. DATA SYNTHESIS: Testing for hemophagocytic lymphohistiocytosis should be initiated promptly in all patients admitted to ICUs with an unexplained or disproportionate inflammatory response, especially those with rapid clinical deterioration. Meeting five or more of eight hemophagocytic lymphohistiocytosis 2004 diagnostic criteria serves as a valuable diagnostic tool for hemophagocytic lymphohistiocytosis. Early aggressive critical care interventions are often required to manage the multisystem organ failure associated with hemophagocytic lymphohistiocytosis. Thorough investigation of the underlying triggers of hemophagocytic lymphohistiocytosis, including infections, malignancies, and autoimmune/autoinflammatory diseases, is essential. Early steroid treatment is indicated for patients with familial hemophagocytic lymphohistiocytosis and is often valuable in patients with acquired hemophagocytic lymphohistiocytosis (i.e., secondary hemophagocytic lymphohistiocytosis) without previous therapy, including macrophage activation syndrome (hemophagocytic lymphohistiocytosis secondary to autoimmune/autoinflammatory disease) without persistent or relapsing disease. Steroid treatment should not be delayed, particularly if organ dysfunction is present. In patients with macrophage activation syndrome, whose disease does not sufficiently respond, interleukin-1 inhibition and/or cyclosporine A is recommended. In familial hemophagocytic lymphohistiocytosis and severe, persistent, or relapsing secondary macrophage activation syndrome, the addition of prompt individualized, age-adjusted etoposide treatment is recommended. CONCLUSIONS: Further studies are needed to determine optimal treatment for patients with hemophagocytic lymphohistiocytosis in ICUs, including the use of novel and adjunct therapies.

Most hereditary forms of hemophagocytic lymphohistiocytosis (HLH) are caused by defects of cytotoxicity, including the vesicle trafficking disorder Griscelli syndrome type 2 (GS2, RAB27A deficiency). Deficiency of the mitogen-activated protein kinase activating death domain protein (MADD) results in a protean syndrome with neurological and endocrinological involvement. MADD acts as a guanine nucleotide exchange factor for small guanosine triphosphatases, including RAB27A. A homozygous splice site mutation in MADD was identified in a female infant with syndromic features, secretory diarrhea, and features of HLH. Aberrant splicing caused by this mutation leads to an in-frame deletion of 30 base pairs and favors other aberrant variants. Patient natural killer (NK) cells and cytotoxic T cells showed a severe degranulation defect leading to absent perforin-mediated cytotoxicity. Platelets displayed defective adenosine triphosphate secretion, similar to that in GS2. To prove causality, we introduced a CRISPR/Cas9-based MADD knockout in the NK cell line NK-92mi. MADD-deficient NK-92mi cells showed a degranulation defect and impaired cytotoxicity similar to that of the patient. The defect of cytotoxicity was confirmed in another patient with MADD deficiency. In conclusion, RAB27A-interacting MADD is involved in vesicle release by cytotoxic cells and platelets. MADD deficiency causes a degranulation defect and represents a novel disease predisposing to an HLH phenotype.

Familial haemophagocytic lymphohistiocytosis (FHL) is an inherited immune deficiency with defective cytotoxicity of natural killer cells and cytotoxic T lymphocytes. A highly stimulated, but ineffective immune response leads to severe hyperinflammation. Clinical and laboratory features are characteristic, but unspecific; thus awareness of FHL is important for early diagnosis. FHL is rapidly fatal without treatment. Standard-of-care therapy is etoposide and corticosteroids, followed by haematopoietic stem cell transplantation (HSCT). CONCLUSION: FHL has become a curable disease with present treatment. Additional cytokine-directed therapy still has to prove its value. Earlier HSCT and less toxic conditioning regimens will lead to improved cure rates.

OBJECTIVE Hyperferritinemia is frequently seen in critically ill patients. A rather rare though life-threatening condition related to severely elevated ferritin is hemophagocytic lymphohistiocytosis. We analyze ferritin levels to differentiate hemophagocytic lymphohistiocytosis from other causes of hyperferritinemia in a mixed cohort of critically ill patients. DESIGN Retrospective observational study. SETTING Adult surgical, anesthesiologic, and medical ICUs of a university hospital. PATIENTS Critical care patients (≥ 18 yr old) admitted to any of the adult ICUs at Charite - Universitatsmedizin Berlin between January 2006 and August 2018 with at least one ferritin value and hyperferritinemia (≥ 500 µg/L). INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Patients were categorized into hemophagocytic lymphohistiocytosis, sepsis, septic shock, and other diagnoses. These were further categorized into 17 subgroups. Hemophagocytic lymphohistiocytosis diagnosis was based on Hemophagocytic Lymphohistiocytosis-2004 criteria and the HScore. Of 2,623 patients with hyperferritinemia, 40 were considered to have hemophagocytic lymphohistiocytosis (1.52%). Maximum ferritin levels were highest in hemophagocytic lymphohistiocytosis patients compared with all other disease groups (each p < 0.001). Sepsis and septic shock patients had higher maximum ferritin levels than patients with other diagnoses (each p < 0.001). A maximum ferritin value of 9,083 µg/L was at 92.5% sensitivity and 91.9% specificity for hemophagocytic lymphohistiocytosis (area under the curve, 0.963; 95% CI, 0.949-0.978). Of all subgroups with other diagnoses, maximum ferritin levels were highest in patients with varicella-zoster virus, hepatitis, or malaria (median, 1,935, 1,928, and 1,587 µg/L, respectively). Maximum ferritin levels were associated with increased in-hospital mortality (odds ratio, 1.518 per log µg/L [95% CI, 1.384-1.665 per log µg/L]; p < 0.001). CONCLUSIONS This is the largest study of patients with ferritin available in a mixed ICU cohort. Ferritin levels in patients with hemophagocytic lymphohistiocytosis, sepsis, septic shock, and other conditions were distinctly different, with the highest ferritin levels observed in hemophagocytic lymphohistiocytosis patients. Maximum ferritin of 9,083 µg/L showed high sensitivity and specificity and, therefore, may contribute to improved diagnosis of hemophagocytic lymphohistiocytosis in ICU. The inclusion of ferritin into the sepsis laboratory panel is warranted.

Background: Elevated serum ferritin is a common condition in critically ill patients. It is well known that hyperferritinemia constitutes a good biomarker for hemophagocytic lymphohistiocytosis (HLH) in critically ill patients. However, further differential diagnoses of hyperferritinemia in adult critically ill patients remain poorly investigated. We sought to systematically investigate hyperferritinemia in adult critically ill patients without HLH. Methods: In this secondary analysis of a retrospective observational study, patients >= 18 years admitted to at least one adult intensive care unit at Charite-Universitatsmedizin Berlin between January 2006 and August 2018, and with hyperferritinemia of >= 500 mu g/L were included. Patients with HLH were excluded. All patients were categorized into non-sepsis, sepsis, and septic shock. They were also classified into 17 disease groups, based on their ICD-10 codes, and pre-existing immunosuppression was determined. Uni- and multivariable linear regression analyses were performed in all patients. Results: A total of 2583 patients were analyzed. Multivariable linear regression analysis revealed positive associations of maximum SOFA score, sepsis or septic shock, liver disease (except hepatitis), and hematological malignancy with maximum ferritin. T/NK cell lymphoma, acute myeloblastic leukemia, Kaposi's sarcoma, acute or subacute liver failure, and hepatic veno-occlusive disease were positively associated with maximum ferritin in post-hoc multivariable linear regression analysis. Conclusions: Sepsis or septic shock, liver disease (except hepatitis) and hematological malignancy are important differential diagnoses in hyperferritinemic adult critically ill patients without HLH. Together with HLH, they complete the quartet of important differential diagnoses of hyperferritinemia in adult critically ill patients. As these conditions are also related to HLH, it is important to apply HLH-2004 criteria for exclusion of HLH in hyperferritinemic patients. Hyperferritinemic critically ill patients without HLH require quick investigation of differential diagnoses.

Background Hemophagocytic lymphohistiocytosis (HLH), an uncontrolled overactivation of the immune system, is well characterized in pediatric patients, yet, much less is known about this life-threatening condition in adult patients. As HLH is often complicated by organ failure, patients will require admission to the intensive care unit for organ support therapy. However, recognition of HLH patients in the intensive care unit (ICU) is challenged by the clinical overlap with sepsis. Here, we analyze HLH patients to better understand its clinical presentation, diagnosis, and treatment. Methods For the purpose of this retrospective observational study, we searched for suspected and diagnosed adult HLH of all patients admitted to at least one adult surgical, anesthesiological or medical ICU between January 2006 and August 2018 at the university hospital Charite - Universitatsmedizin Berlin. All cases were reviewed by two HLH experts, who confirmed or declined the diagnosis. Results Of 6,340 ICU patients with ferritin measurement, 40 suffered from HLH (0.63%). Of these, in-hospital mortality was 60.0% over all cases, which was highest in malignancy-associated HLH (71.4%). Infections were identified as most common triggers (42.5%). A variety of 19 different treatment strategies were applied. Non-survivors showed higher ferritin at diagnosis compared with survivors (P = 0.021), which was also seen in multivariable analyses. A minimum ferritin of 4083 μg/L after diagnosis was most predictive for 30-day mortality (AUC 0.888, 95% CI 0.771-1.000; sensitivity 93.8%, specificity 78.9%). Conclusions Mortality in adult HLH patients in the ICU is high, particularly in malignancy-associated HLH. Infections are the most frequent HLH triggers in critically ill patients. At present, there is no standardized treatment for HLH in adult patients available. Assessment of ferritin is valuable for diagnosis, prognosis, and treatment monitoring. Trial registration The study was registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016.

Background Ferritin is the major iron storage protein and an acute phase reactant. Hyperferritinemia is frequently seen in the critically ill where it has been hypothesized that not only underlying conditions but also factors such as transfusions, hemodialysis and extracorporeal life support (ECLS) lead to hyperferritinemia. This study aims to investigate the influence of transfusions, hemodialysis, and ECLS on hyperferritinemia in a multidisciplinary ICU cohort. Methods This is a post-hoc analysis of a retrospective observational study including patients aged ≥ 18 years who were admitted to at least one adult ICU between January 2006 and August 2018 with hyperferritinemia ≥ 500 μg/L and of ≥ 14 days between two ICU ferritin measurements. Patients with hemophagocytic lymphohistiocytosis (HLH) were excluded. To identify the influence of transfusions, hemodialysis, and ECLS on ferritin change, multivariable linear regression analysis with ferritin change between two measurements as dependent variable was performed. Results A total of 268 patients was analyzed. Median duration between measurements was 36 days (22–57). Over all patients, ferritin significantly increased between the first and last measurement (p = 0.006). Multivariable linear regression analysis showed no effect of transfusions, hemodialysis, or ECLS on ferritin change. Changes in aspartate aminotransferase (ASAT) and sequential organ failure assessment (SOFA) score were identified as influencing factors on ferritin change [unstandardized regression coefficient (B) = 2.6; (95% confidence interval (CI) 1.9, 3.3); p < 0.001 and B = 376.5; (95% CI 113.8, 639.1); p = 0.005, respectively]. Using the same model for subgroups of SOFA score, we found SOFA platelet count to be associated with ferritin change [B = 1729.3; (95% CI 466.8, 2991.9); p = 0.007]. No association of ferritin change and in-hospital mortality was seen in multivariable analysis. Conclusions The present study demonstrates that transfusions, hemodialysis, and ECLS had no influence on ferritin increases in critically ill patients. Hyperferritinemia appears to be less the result of iatrogenic influences in the ICU thereby underscoring its unskewed diagnostic value. Trial registration The study was registered with www.ClinicalTrials.gov (NCT02854943) on August 1, 2016.

OBJECTIVES Hemophagocytic lymphohistiocytosis is a cytokine release syndrome caused by uncontrolled immune activation resulting in multiple organ failure and death. In this systematic review, we aimed to analyze triggers, various treatment modalities, and mortality in critically ill adult hemophagocytic lymphohistiocytosis patients. DATA SOURCES MEDLINE database (PubMed) at October 20, 2019. STUDY SELECTION Studies and case series of patients greater than or equal to 18 years old, of whom at least one had to be diagnosed with hemophagocytic lymphohistiocytosis and admitted to an ICU. DATA EXTRACTION Source data of studies and case series were summarized and analyzed on an individual basis. Multivariable logistic regression analysis was performed adjusting for age, sex, and trigger groups. Each single treatment agent was entered as a dichotomous variable to determine treatments associated with survival, regardless if given alone or in combination. DATA SYNTHESIS In total, 661 patients from 65 studies and case series were included. Overall mortality was 57.8%. Infections were the most frequent trigger (49.9%), followed by malignancies (28.0%), autoimmune diseases (12.1%), unknown triggers (9.4%), and drugs (0.6%). Treatment with IV immunoglobulins was associated with improved survival (odds ratio, 0.548; 95% CI, 0.337-0.891; p = 0.015), while treatment with cyclosporine was associated with increased risk of death (odds ratio, 7.571; 95% CI, 3.702-15.483; p < 0.001). Considering different trigger groups separately, same results occurred only for infection-triggered hemophagocytic lymphohistiocytosis. No information was available on disease severity and other confounding factors. CONCLUSIONS Mortality of hemophagocytic lymphohistiocytosis in the ICU is high. Most common triggers were infections. Results of survival analyses may be biased by treatment indication and disease severity. Future studies prospectively investigating treatment tailored to critically ill hemophagocytic lymphohistiocytosis patients are highly warranted.

BACKGROUND Reduced toxicity conditioning for hematopoietic stem cell transplantation of patients with hemophagocyticlymphohistiocytosis (HLH) results in favorable survival, however at the expense of relevant rates of mixed chimerism. Factors predisposing to mixed chimerism remain to be determined. PROCEDURE Patients with primary HLH transplanted 2009-2016 after treosulfan- or melphalan-based conditioning regimens were analyzed in a retrospective multicenter study for survival, engraftment, chimerism, and adverse events. Mixed chimerism was considered substantial if

We report the largest prospective study thus far on hematopoietic stem cell transplantation (HSCT) in hemophagocytic lymphohistiocytosis (HLH), a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. Although all patients with HLH typically need intensive anti-inflammatory therapy, patients with FHL also need HSCT to be cured. In the international HLH-2004 study, 187 children aged

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome requiring aggressive immunosuppressive therapy. Following 2 large international studies mainly targeting pediatric patients with familial disease and patients without underlying chronic or malignant disease, the HLH-94 protocol is recommended as the standard of care when using etoposide-based therapy by the Histiocyte Society. However, in clinical practice, etoposide-based therapy has been widely used beyond the study inclusion criteria, including older patients and patients with underlying diseases (secondary HLH). Many questions remain around these extended indications and published reports do not address several practical issues. To tackle these concerns, the HLH Steering Committee of the Histiocyte Society decided to issue guidance for use of the HLH-94 protocol. The group convened in a structured consensus finding process to define recommendations that are based largely on expert opinion backed up by available data from the literature. The recommendations address all main elements of HLH-94 including corticosteroids, cyclosporin, etoposide, intrathecal therapy, and hematopoietic stem cell transplantation (HSCT) and consider various forms of HLH and all age groups. Aspects covered include indications, applications, dosing, side effects, duration of therapy, salvage therapy, and HSCT. These recommendations aim to provide a framework to guide treatment decisions in this severe disease.

Hemophagocytic lymphohistiocytosis (HLH) is a life‐threatening inflammatory syndrome characterized by hyperactivation of lymphocytes and histiocytes. T cells play a key role in HLH pathogenesis, but their differentiation pattern is not well characterized in patients with active HLH. We compared T‐cell activation patterns between patients with familial HLH (1°HLH), 2°HLH without apparent infectious trigger (2°HLH) and 2°HLH induced by a viral infection (2°V‐HLH). Polyclonal CD8+ T cells are highly activated in 1°HLH and 2°V‐HLH, but less in 2°HLH as assessed by HLA‐DR expression and marker combination with CD45RA, CCR7, CD127, PD‐1 and CD57. Absence of increased HLA‐DR expression on T cells excluded active 1° HLH with high sensitivity and specificity. A high proportion of polyclonal CD127−CD4+ T cells expressing HLA‐DR, CD57, and perforin is a signature of infants with 1°HLH, much less prominent in virus‐associated 2°HLH. The similar pattern and extent of CD8+ T‐cell activation compared to 2° V‐HLH is compatible with a viral trigger of 1°HLH. However, in most 1°HLH patients no triggering infection was documented and the unique activation of cytotoxic CD4+ T cells indicates that the overall T‐cell response in 1°HLH is different. This may reflect different pathways of pathogenesis of these two HLH variants., The illustrated phenotype is characteristic for patients presenting with different subtypes of HLH in the first year of life and demonstrates the pathophysiological principle. In older patients, most markers of T cell differentiation (apart from HLA‐DR) are very heterogenous also in healthy individuals, such that these phenotypic markers are of limited diagnostic value in patients beyond infancy.

We conducted a clinical trial and report the long-term outcome of 773 children with acute lymphoblastic leukemia upon risk-adapted therapy accrued in trial CoALL 07-03 (from the Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia). In a 2-step stratification, patients were allocated to receive either low- or high-risk treatment, based on initial white blood cell count, age, and immunophenotype. A second stratification was performed according to the results of in vitro pharmacosensitivity toward prednisolone, vincristine, and asparaginase (PVA score) and in vivo response after induction therapy (minimal residual disease [MRD]). Therapy was reduced for both risk groups in patients with a low PVA score or negative MRD result, and intensified in patients with a high PVA score. Overall outcome improved significantly compared with the predecessor CoALL 06-97 trial, with identical therapy backbone despite treatment reduction in 15.8% of patients (10-year probability of event-free survival, 83.5% vs 73.9%; overall survival, 90.7% vs 83.8%). Outcome for patients in the reduced treatment arms was superior to that of patients in the standard arms, associated with a profound reduction in frequency and severity of infectious complications. Importantly, we observed a lack of correlation between in vitro and in vivo drug response, as well as a lower predictive value of in vitro drug testing, reflecting an intrinsic limitation of this methodology that prevents its use for treatment stratification in future trials. In conclusion, it might be possible to reduce chemotherapy in children with acute lymphoblastic leukemia selected by stringent in vivo measurement of MRD without jeopardizing overall outcome.

Item does not contain fulltext Patient registries are instrumental for clinical research in rare diseases. They help to achieve a sufficient sample size for epidemiological and clinical research and to assess the feasibility of clinical trials. The European Society for Immunodeficiencies (ESID) registry currently comprises information on more than 25,000 patients with inborn errors of immunity (IEI). The prerequisite of a patient to be included into the ESID registry is an IEI either defined by a defect in a gene included in the disease classification of the international union of immunological societies, or verified by applying clinical criteria. Because a relevant number of patients, including those with common variable immunodeficiency (CVID), representing the largest group of patients in the registry, remain without a genetic diagnosis, consensus on classification of these patients is mandatory. Here, we present clinical criteria for a large number of IEI that were designed in expert panels with an external review. They were implemented for novel entries and verification of existing data sets from 2014, yielding a substantial refinement. For instance, 8% of adults and 27% of children with CVID (176 of 1704 patients) were reclassified to 22 different immunodeficiencies, illustrating progress in genetics, but also the previous lack of standardized disease definitions. Importantly, apart from registry purposes, the clinical criteria are also helpful to support treatment decisions in the absence of a genetic diagnosis or in patients with variants of unknown significance.

Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome induced by aberrantly activated macrophages and cytotoxic T cells. The primary (genetic) form, caused by mutations affecting lymphocyte cytotoxicity and immune regulation, is most common in children, whereas the secondary (acquired) form is most frequent in adults. Secondary HLH is commonly triggered by infections or malignancies but may also be induced by autoinflammatory/autoimmune disorders, in which case it is called macrophage activation syndrome (MAS; or MAS-HLH). Most information on the diagnosis and treatment of HLH comes from the pediatric literature. Although helpful in some adult cases, this raises several challenges. For example, the HLH-2004 diagnostic criteria developed for children are commonly applied but are not validated for adults. Another challenge in HLH diagnosis is that patients may present with a phenotype indistinguishable from sepsis or multiple organ dysfunction syndrome. Treatment algorithms targeting hyperinflammation are frequently based on pediatric protocols, such as HLH-94 and HLH-2004, which may result in overtreatment and unnecessary toxicity in adults. Therefore, dose reductions, individualized tailoring of treatment duration, and an age-dependent modified diagnostic approach are to be considered. Here, we present expert opinions derived from an interdisciplinary working group on adult HLH, sponsored by the Histiocyte Society, to facilitate knowledge transfer between physicians caring for pediatric and adult patients with HLH, with the aim to improve the outcome for adult patients affected by HLH.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune dysregulation disease. It can be subdivided into a primary, genetic form and a secondary form that complicates diverse infections, autoimmune or autoinflammatory disorders, and malignancies. Both forms, primary and secondary HLH, present with the same spectrum of non-specific symptoms, making accurate diagnosis and rapid treatment initiation challenging. A prompt diagnosis is crucial for appropriate therapeutic management. Clinical diagnosis of an HLH episode, followed by sequential immunological testing, and genetic diagnosis provide the basis for a rapid decision on hematopoietic stem cell transplantation indicated in patients with primary HLH.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. In the HLH-94 study, with an estimated 5-year probability of survival (pSu) of 54% (95% confidence interval, 48%-60%), systemic therapy included etoposide, dexamethasone, and, from week 9, cyclosporine A (CSA). Hematopoietic stem cell transplantation (HSCT) was indicated in patients with familial/genetic, relapsing, or severe/persistent disease. In HLH-2004, CSA was instead administered upfront, aiming to reduce pre-HSCT mortality and morbidity. From 2004 to 2011, 369 children aged

We report our experience in using flow cytometry-based immunological screening prospectively as a decision tool for the use of genetic studies in the diagnostic approach to patients with hemophagocytic lymphohistiocytosis (HLH). We restricted genetic analysis largely to patients with abnormal immunological screening, but included whole exome sequencing (WES) for those with normal findings upon Sanger sequencing. Among 290 children with suspected HLH analyzed between 2010 and 2014 (including 17 affected, but asymptomatic siblings), 87/162 patients with "full" HLH and 79/111 patients with "incomplete/atypical" HLH had normal immunological screening results. In 10 patients, degranulation could not be tested. Among the 166 patients with normal screening, genetic analysis was not performed in 107 (all with uneventful follow-up), while 154 single gene tests by Sanger sequencing in the remaining 59 patients only identified a single atypical CHS patient. Flow cytometry correctly predicted all 29 patients with FHL-2, XLP1 or 2. Among 85 patients with defective NK degranulation (including 13 asymptomatic siblings), 70 were Sanger sequenced resulting in a genetic diagnosis in 55 (79%). Eight patients underwent WES, revealing mutations in two known and one unknown cytotoxicity genes and one metabolic disease. FHL3 was the most frequent genetic diagnosis. Immunological screening provided an excellent decision tool for the need and depth of genetic analysis of HLH patients and provided functionally relevant information for rapid patient classification, contributing to a significant reduction in the time from diagnosis to transplantation in recent years.

OBJECTIVE: To develop and validate a diagnostic score that assists in discriminating primary hemophagocytic lymphohistiocytosis (pHLH) from macrophage activation syndrome (MAS) related to systemic juvenile idiopathic arthritis. STUDY DESIGN: The clinical, laboratory, and histopathologic features of 362 patients with MAS and 258 patients with pHLH were collected in a multinational collaborative study. Eighty percent of the population was assessed to develop the score and the remaining 20% constituted the validation sample. Variables that entered the best fitted model of logistic regression were assigned a score, based on their statistical weight. The MAS/HLH (MH) score was made up with the individual scores of selected variables. The cutoff in the MH score that discriminated pHLH from MAS best was calculated by means of receiver operating characteristic curve analysis. Score performance was examined in both developmental and validation samples. RESULTS: Six variables composed the MH score: age at onset, neutrophil count, fibrinogen, splenomegaly, platelet count, and hemoglobin. The MH score ranged from 0 to 123, and its median value was 97 (1st-3rd quartile 75-123) and 12 (1st-3rd quartile 11-34) in pHLH and MAS, respectively. The probability of a diagnosis of pHLH ranged from 99% for a score of =123. A cutoff value of =60 revealed the best performance in discriminating pHLH from MAS. CONCLUSION: The MH score is a powerful tool that may aid practitioners to identify patients who are more likely to have pHLH and, thus, could be prioritized for functional and genetic testing.

Opinion statement Central nervous system (CNS)-hemophagocytic lymphohistiocytosis (HLH) is not a disease in itself, but it is part of a systemic immune response. The vast majority of patients with CNS-HLH also have systemic HLH and a large number of patients with primary and secondary HLH have CNS involvement. Reactivations within the CNS are frequent during the course of HLH treatment and may occur concomitant with or independent of systemic relapses. It is also important to consider primary HLH as an underlying cause of “unknown CNS inflammation” as these patients may present with only CNS disease. To initiate proper treatment, a correct diagnosis must be made. A careful review of the patient’s history and a thorough neurological examination are essential. In addition to the blood tests required to make a diagnosis of HLH, a lumbar puncture with cerebrospinal fluid (CSF) analysis and magnetic resonance imaging (MRI) should always be done in all cases regardless of the presence or absence of neurological signs or symptom. Treatment options for CNS-HLH include, but are not limited to, those commonly used in systemic HLH, including corticosteroids, etoposide, cyclosporine A, alemtuzumab, and ATG. In addition, intrathecal treatment with methotrexate and corticosteroids has become a standard care and is likely to be beneficial. Therapy must be initiated without inappropriate delay to prevent late effects in HLH. An interesting novel approach is an anti-IFN-gamma antibody (NI-0501), which is currently being tested. Hematopoietic stem cell transplantation (HSCT) also represents an important CNS-HLH treatment; patients with primary HLH may benefit from immediate HSCT even if there is active disease at time of transplantation, though care should be taken to monitor CNS inflammation through HSCT and treat if needed. Since CNS-HLH is a condition leading to the most severe late effects of HLH, early expert consultation is recommended.

In hematopoietic stem cell transplantation for hemophagocytic lymphohistiocytosis, high transplant-related mortality after busulfan-based myeloablative regimens has been observed. Conditioning regimens with reduced toxicity based on melphalan or treosulfan are promising alternatives. We retrospectively analyzed hematopoietic stem cell transplantations in 19 hemophagocytic lymphohistiocytosis patients after conditioning with fludarabine, treosulfan, alemtuzumab, with or without thiotepa. Overall and disease-free survivals were 100% (follow up 7–31 months). Two patients required second transplant (1 after haploidentical transplantation). In 6 patients, overall donor chimerism dropped below 75% and prompted donor lymphocyte infusions. Administration of donor lymphocytes or second transplantation were significantly more frequent after transplantation from a human leukocyte antigen mismatched (9/10) versus matched (10/10) donor (P=0.018). The toxicity profile was favorable, with one veno-occlusive disease, one grade 3 graft-versus-host disease after donor lymphocyte infusion, and 2 severe viral infections (1 influenza, 1 Epstein Barr virus). In conclusion, the treosulfan-based regimen in hemophagocytic lymphohistiocytosis is effective with low toxicity and gives excellent overall and disease-free survival rates. In the future, the incidence of mixed chimerism, particularly after human leukocyte antigen mismatched donor transplants, needs to be addressed.

Syntaxin-11 (Stx11), an atypical member of the SNARE protein family, is part of the cytolytic machinery of T and NK cells and involved in the fusion of lytic granules with the plasmamembrane. Functional loss of syntaxin-11 in humans causes defective degranulation and impaired cytolytic activity of T and NK cells. Furthermore, patients with STX11 deficiency develop familial hemophagocytic lymphohistiocytosis type 4 (FHL4), a life-threatening disease of severe hyperinflammation. We established Stx11-deficient mice as an animal model for FHL4. Stx11-deficient mice exhibited severely reduced degranulation and cytolytic activity of CTL and NK cells and developed all clinical symptoms of hemophagocytic lymphohistiocytosis (HLH) after infection with lymphocytic choriomeningitis virus (LCMV). The HLH phenotype was further characterized by hyperactive CD8 T cells and continuous IFN-γ production. However, in contrast to perforin-deficient mice, which represent a model for FHL2, progression of HLH was not fatal. Survival of Stx11-deficient mice was determined by exhaustion of antigen-specific T cells, characterized by expression of inhibitory receptors, sequential loss of effector functions, and finally T-cell deletion. Blockade of inhibitory receptors on T cells in Stx11-deficient mice converted nonfatal disease course into fatal HLH, identifying T-cell exhaustion as an important factor for determination of disease severity in HLH.

Differential diagnosis of hemophagocytic lymphohistiocytosis (HLH; hemophagocytic syndrome) and sepsis is critically important because the life-saving aggressive immunosuppressive treatment, required in the effective HLH therapy, is absent in sepsis guidelines. Moreover, HLH may be complicated by sepsis. Hyperinflammation, present in both states, gives an overlapping clinical picture including fever and performance status deterioration. The aim of this review is to provide aid in this challenging diagnostic process. Analysis of clinical features and laboratory results in multiple groups of patients (both adult and pediatric) with either HLH or sepsis allows to propose criteria differentiating these two conditions. The diagnosis of HLH is supported by hyperferritinemia, splenomegaly, marked cytopenias, hypofibrinogenemia, low CRP, characteristic cytokine profile and, only in adults, hypertriglyceridemia. In the presence of these parameters (especially the most characteristic hyperferritinemia), the other HLH criteria should be assessed. Genetic analyses can reveal familial HLH. Hemophagocytosis is neither specific nor sensitive for HLH.

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Two related boys who died from fulminant infectious mononucleosis were diagnosed with X-linked lymphoproliferative disease type 1 (XLP-1). Family screening (n=17) identified 6 female mutation carriers and 2 more XLP-1 patients in whom, despite recurrent infections, agammaglobulinemia, and Hodgkin's Disease, the genetic basis had been unknown; demonstrating that awareness and early genetic testing are crucial to reveal underlying primary immunodeficiencies and improve outcome. Furthermore, XLP should be included routinely in the differential diagnosis of severe hypogammaglobulinemia and/or lymphoma in males.

These guidelines for the management of patients up to 18 years with Langerhans cell histiocytosis (LCH) have been set up by a group of experts involved in the Euro Histio Net project who participated in national or international studies and in peer reviewed publications. Existing guidelines were reviewed and changed where new evidence was available in the literature up to 2012. Data and publications have been ranked according to evidence based medicine and when there was a lack of published data, consensus between experts was sought. Guidelines for diagnosis, initial clinical work-up, and treatment and long-term follow-up of LCH patients are presented.

In Germany, nearly every child afflicted by a malignant dis-ease or a central nervous system tumor is enrolled in a clinicaltrial during treatment. Many of these children are under the age of5 years when they are first diagnosed [1]. The decision whether ornot to participate in a clinical trial is normally made by theparents on behalf of their child. Therefore, ensuring adequatelyinformed parental consent is essential to ethical practice in pedi-atric oncology.However, many empirical studies have revealed difficultieswith the informed consent process when parents are faced withthe decision to enroll their child in a clinical trial. Previous studyfindings have indicated that recalling signing the consent docu-ment and understanding the information given about the plannedresearch are particular challenges of the consent process. Parentsmay, for example, mistakenly understand that the research projectwas designed to benefit their child directly [2]. They may alsomisunderstand key concepts of research designs such as randomi-zation [3,4], or they may even be unaware that they have giventheir consent to enroll their child in a clinical trial [5].Confronted with a severe diagnosis, parents’ understandingand memory are influenced by high levels of emotional distressat the time they are asked for consent [6–9]. Although parentsmay receive appropriate information about the purposes, risks,and benefits of a clinical trial, they may not be able to understandor recall it correctly. In this context, the impact of education andmigration is discussed in the literature [10–15]. There is someempirical evidence that parents with low educational status andthose belonging to minority groups are at greater risk than othersof enrolling their child in a clinical trial without fully understand-ing the implications [13,15]. However, common pediatric cancerssuch as acute leukemia require immediate treatment and an im-mediate parental decision as to whether or not their child shouldbe enrolled in a clinical trial [16].As the existing empirical studies have mainly been conductedin the United States and Canada, we examined the process ofparental consent in pediatric cancer trials in Germany. We presenthere the first population-based survey on parents’ memory andexperience of the consent process. In order to evaluate parents’perception and recollection of the written consent process, wewanted to know how well the decision regarding enrollmentwas stored in parents’ memory. Therefore, we first asked theparents whether or not they had consented to their child’s partici-pation in a clinical trial. If the answer was yes, we wanted toknow how well they remembered the informed consent processitself; we then evaluated the parents’ subjective satisfaction withthe consent process by asking whether they felt adequately in-formed at the time they made their decision.

Background The anthracyclines daunorubicin (DNR) and doxorubicin (DOX) are among the most important drugs in the treatment of childhood acute lymphoblastic leukemia, however there are conflicting in vitro data about the comparative efficacy and equivalent doses of both anthracyclines. To address the question of in vivo efficacy of both anthracyclines, patients enrolled in the CoALL 07-03 trial were randomized to receive one single dose of either doxorubicin 30 mg/m2, daunorubicin 30 mg/m2, or daunorubicin 40 mg/m2 upfront induction therapy. Procedure Children with newly diagnosed B-Precursor ALL or T-ALL were eligible for the randomized comparison. From the percentage of blasts and the white blood cell count (WBC) the absolute number of leukemic cells per µl peripheral blood (PB) was calculated and the initial value before DOX/DNR infusion equated as 100%. Main target criterion of this study was the leukemic cell decrease from Day 0 to Day 7. Results Seven hundred forty three patients were randomized: 247 to the DOX; 252 to the DNR 30 mg/m2; and DNR to the 40 mg/m2 arm. The in vivo response was similar in all three treatment arms with a comparable blast decline in the peripheral blood. The percentages of patients with a clear non-response (M3 marrow) and moreover, the level of minimal residual disease (MRD) on Day 15 or at the end of induction were similar. Conclusion In vivo efficacy of a single dose daunorubicin 30 or 40 mg/m2 is similar to that of doxorubicin given in a dose of 30 mg/m2. Pediatr Blood Cancer 2013;60:254–257. © 2012 Wiley Periodicals, Inc.

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of severe hyperinflammation caused by the uncontrolled proliferation of activated lymphocytes and histiocytes secreting high amounts of inflammatory cytokines. Cardinal signs and symptoms are prolonged fever, hepatosplenomegaly and pancytopenia. Characteristic biochemical markers include elevated triglycerides, ferritin and low fibrinogen. HLH occurs on the basis of various inherited or acquired immune deficiencies. Impaired function of natural killer (NK) cells and cytotoxic T-cells (CTL) is shared by all forms of HLH. Genetic HLH occurs in familial forms (FHLH) in which HLH is the primary and only manifestation, and in association with the immune deficiencies Chédiak-Higashi syndrome 1 (CHS 1), Griscelli syndrome 2 (GS 2) and x-linked lymphoproliferative syndrome (XLP), in which HLH is a sporadic event. Most patients with acquired HLH have no known underlying immune deficiency. Both acquired and genetic forms are triggered by infections, mostly viral, or other stimuli. HLH also occurs as a complication of rheumatic diseases (macrophage activation syndrome) and of malignancies. Several genetic defects causing FHLH have recently been discovered and have elucidated the pathophysiology of HLH. The immediate aim of therapy in genetic and acquired HLH is suppression of the severe hyperinflammation, which can be achieved with immunosuppressive/immunomodulatary agents and cytostatic drugs. Patients with genetic forms have to undergo stem cell transplantation to exchange the defective immune system with normally functioning immune effector cells. In conclusion, awareness of the clinical symptoms and of the diagnostic criteria of HLH is crucial in order not to overlook HLH and to start life-saving therapy in time.

Aim: Haemophagocytic lymphohistiocytosis (HLH) is a life-threatening disturbance of immunoregulation. HLH comprises primary and acquired forms with different disease severity. A large proportion of deaths occur early into treatment. We investigated association with early death for laboratory and clinical parameters before the start of and 2 weeks into therapy. Methods: A total of 232 children from Scandinavia, Germany or Italy, fulfilling diagnostic criteria and/or with familial disease and/or HLH-causing mutations, receiving HLH treatment 1994–2008 were included. The relation between clinical findings and early pre-transplant death was examined using the Cox proportional hazards model, with a 4-month right-truncation of the outcome. Patients were censored at last follow-up or transplant. Statistically significant predictors were adjusted for sex, age and each other. Results: The following features were significantly associated with adverse outcome: hyperbilirubinaemia (>50 μmol/L; adjusted hazard ratio (aHR) 3.2; 95% confidence interval 1.3–8.1, p = 0.011), hyperferritinaemia (>2000 μg/L; aHR 3.2; 1.2–8.6, p = 0.019), cerebrospinal fluid pleocytosis (>100 × 106/L; aHR 5.1; 1.4–18.5, p = 0.012) at diagnosis, and thrombocytopenia ( 2000 μg/L; aHR 10.6; 1.2–96.4, p = 0.037) 2 weeks into therapy. Non-improvement of fever, anaemia and/or thrombocytopenia also had adverse impact. Conclusion: There seem to be easily available clinical predictors of early mortality in HLH patients, which may help guide treatment decisions.

Children with chromosomal instability syndromes have an increased risk of developing lymphoma and leukaemia. The treatment of these malignancies is hampered by therapy-associated toxicity and infectious complications. This retrospective analysis evaluated the therapy outcome of 38 children with Ataxia teleangiectasia or Nijmegen-breakage syndrome with acute lymphoblastic leukaemia (ALL, n = 9), Non-Hodgkin lymphoma (NHL, n = 28) and Hodgkin lymphoma (HL, n = 1). All patients with NHL or ALL were treated in accordance to Berlin-Frankfurt-Munster (BFM)- or Co-operative study group for childhood ALL (CoALL)-oriented chemotherapy schedules. 22 patients received significantly reduced-intensity chemotherapy. After a median follow-up of 3·7 years the 10-year overall survival was 58%. Dosage-reduction of chemotherapeutic drugs seemed to have no disadvantages and reduced toxic side effects. On the other hand, reduced-intensity chemotherapy did not prevent second malignancies, which occurred in ten patients with a 10-year incidence of 25%. After individual treatment approaches three of these patients with second malignancies were in complete clinical remission for more than 5 years. We conclude that BFM- or CoALL-oriented chemotherapy is effective and can be administered in children with AT or NBS. Moreover, we show that even second lymphoid malignancies can successfully be treated in these patients.

Background Mutations of UNC13D are causative for familial haemophagocytic lymphohistiocytosis type 3 (FHL3; OMIM 608898). Objective To carry out a genotype–phenotype study of patients with FHL3. Methods A consortium of three countries pooled data on presenting features and mutations from individual patients with biallelic UNC13D mutations in a common database. Results 84 patients with FHL3 (median age 4.1 months) were reported from Florence, Italy (n=54), Hamburg, Germany (n=18), Stockholm, Sweden (n=12). Their ethnic origin was Caucasian (n=57), Turkish (n=10), Asian (n=7), Hispanic (n=4), African (n=3) (not reported (n=3)). Thrombocytopenia was present in 94%, splenomegaly in 96%, fever in 89%. The central nervous system (CNS) was involved in 49/81 (60%) patients versus 36% in patients with FHL2 (p=0.001). A combination of fever, splenomegaly, thrombocytopenia and hyperferritinaemia was present in 71%. CD107a expression, NK activity and Munc 13-4 protein expression were absent or reduced in all but one of the evaluated patients. 54 different mutations were observed, including 15 new ones: 19 missense, 14 deletions or insertions, 12 nonsense, nine splice errors. None was specific for ethnic groups. Patients with two disruptive mutations were younger than patients with two missense mutations (p

In a study of childhood acute lymphoblastic leukemia (CoALL 06-97 study), the in vitro sensitivity of the patients' cells to prednisolone, vincristine and asparaginase was introduced as a new additional risk parameter for treatment stratification. In parallel in vivo treatment response was assessed by determining the presence and extent of minimal residual disease in a subset of patients (n=224). Here we report the long-term impact of in vitro sensitivity-based risk stratification according to survival and compare the results of in vitro sensitivity with in vivo response.Patients with a sensitive in vitro profile were treated with a reduced intensity protocol (n=167) whereas patients defined as low risk according to conventional parameters but with a resistant in vitro profile were given intensified therapy (n=47).At a median follow-up of 6.8 years event-free survival was 0.80±0.03 for patients with a sensitive profile, 0.73±0.03 for those with an intermediate profile and 0.67±0.08 for those with a resistant profile (P=0.015). Overall, the treatment results of the cases stratified according to in vitro sensitivity were similar to those of the historical control group stratified based on conventional risk factors. Minimal residual disease at the end of induction was a strong predictor of outcome in B-precursor and T-cell acute lymphoblastic leukemia. There was no correlation between in vitro and in vivo treatment response in B-precursor leukemia (Spearman's r=0.13; P=0.15) in contrast to T-cell acute lymphoblastic leukemia (Spearman's r=0.63; P0.001)A moderate reduction in treatment intensity for patients with a sensitive in vitro profile was possible without jeopardizing treatment outcome. However, in vitro drug testing was affected by a decrease in risk predictive power over time and was not correlated with in vivo assessment of minimal residual disease in B-precursor acute lymphoblastic leukemia. It was, therefore, abandoned in favor of the assessment of in vivo response in subsequent CoALL trials.

Hyperferritinemia is a hallmark of hemophagocytic lymphohistiocytosis (HLH). In the HLH-2004 criteria, a ferritin value >500 µg/L is considered positive. However, this level was not determined based on evidence. We compared 123 patients with HLH and 320 patients with other hyperferritinemic conditions, calculated a receiver-operating characteristic, and determined sensitivity and specificity for different values. At 2,000 µg/L a trade-off is reached with sensitivity at 70% and specificity at 68%. If familial HLH and virus-associated acquired HLH are analyzed separately, sensitivity and specificity are similar for this level. The results may guide a potential modification of the current HLH criteria.

Juvenile xanthogranuloma (JXG) is a disorder of disputed origin thought to be related to the dermal/interstitial macrophage. A 5-year-old female presented with an aggressive systemic JXG that developed 5 months after the diagnosis of T-cell acute lymphoblastic leukemia (T-ALL). Examination of the T-cell receptor gamma (TCR-γ) rearrangement in T-ALL blasts, JXG infiltrated lymph node biopsies and micro-dissected JXG histiocytes revealed an identical bi-allelic TCR-γ rearrangement in all samples, thus providing evidence for a clonal relationship between T-ALL and JXG in this case.

In this report, we evaluate the hypothesis that hemophagocytic lymphohistiocytosis in patients with defects of lymphocyte cytotoxicity is usually triggered by infections. We show that in the majority of patients, extensive virus PCR panels performed in addition to routine microbiological investigations remain negative and summarize 25 patients with onset of hemophagocytic lymphohistiocytosis in utero or within the first 10 days of life, in none of which an associated bacterial or viral infection was reported. These observations, even though preliminary, invite to consider a key role of lymphocyte cytotoxicity in controlling T‐cell homeostasis also in the absence of apparent infectious stimuli.